Abstract
The Philadelphia chromosome (Ph1), detected in virtually all cases of chronic myelogenous leukemia (CML), is formed by a reciprocal translocation between chromosome 9 and 22 that fuses Bcr-encoded sequences upstream of exon 2 of c-Abl. This oncogene produces a fusion protein, p210bcr-abl, in which the Abl tyrosine kinase activity is elevated. Using anti-phosphotyrosine immunoblotting, we have compared the pattern of phosphotyrosine-containing proteins from freshly prepared neutrophils of patients in the stable phase of CML to normal controls. The only consistent difference was the presence of a 39-kDa tyrosine-phosphorylated protein in 18 out of 18 neutrophil samples from CML patients that was not seen in normal controls. This same protein, as assessed by two-dimensional anti-phosphotyrosine immunoblotting, was also present in cell lines expressing p210bcr-abl, including K562 cells. Using K562 cells as a source of protein, the 39-kDa protein was purified and identified by microsequencing as Crkl, an SH2/SH3 adaptor protein related to the crk oncogene of the avian sarcoma virus, CT10. A direct interaction between Crkl and Abl has also been shown using a yeast two-hybrid screen.
Highlights
From the wivision of Hematology a n d Medical Oncology, Oregon Health Sciences University, Portland, Oregon 97201 a n d the Divisions of $Cellular a n d Molecular Biology a n d Wematologic Malignancies, Dana-Farber Cancer Znstitute a n d Harvard Medical School, Boston, Massachusetts 02115 kinase relevant forthe stable phase of CML, we have examined stable phase neutrophils for evidence of novel tyrosine-phosphorylated proteins and have identified a single cellular protein, p39, that is universally and uniquely tyrosine-phosphorylated in CML neutrophils
Using K562 cells as a source of protein, the 39-kDa protein was purifiedand identified by microsequencingas Crkl, an istasmsuticorToatfensoilhavolryemm imptayPreeloohldi9tsfcieblaaaeieynsnlxdee,odevsanlpapo22rt2fh2ee1odicca0fti.hhpbcUcrarc-hootAsrnri-cinfbo"niaucgm lbl.sw'etamoT,rhsnsahyiotBcneiim-hslscpolreoohg-tnceeho(annPcsetpAoochioguhbo'1de)sno,nettbydldeyereeruposttokweesrsceiqoinenm tudeeeeeundiaincimkcncehai(esmnCvsrfaiouM urus----epL-)iasa,sSsucvtHirgliniea2gkgen/eeSnlsssyH,ota.iCm3nrTcrgeakhotdloemhfaswaaepttahtvdsoaeiarradtbuplaiissroro,esoluocCtigtgedTiigeclnieOnnesttrfite(eff1treilhaea5ccadt,testt1aidooC6sfn)tra.poknU2bl1tAesih0sitbebnwaclg'-ce"cibreanak'n.tnyoederntaiahcdcsoeattsgitneteewgnfteoopw-rrhoooyfmtbepteirhrndiode,it-eins noblotting, we have compared the pattern of phospho
Ence of a 39-kDa tyrosine-phosphorylated proteinin 18 Neutrophils were prepared, stimulated with cytokines as indicated, out of18 neutrophil samples fromCML patients that wasand lysed as described [17].Purity was typically >98%as assessed by not seen in normal controls
Summary
Tyrosine-containing trophils of patients ipnrottheeinfsrstoamblferepshholafysCeMprLetpoarneodrmnaelu-froPmrefpreasrhat,ihoenpaorfinHizuemd balnooNd eduratrwopnhfirlosm-NpeuattrieonpthsilwswitehrePh'(+)prCeMpaLreidn controls. Ence of a 39-kDa tyrosine-phosphorylated proteinin 18 Neutrophils were prepared, stimulated with cytokines as indicated, out of neutrophil samples fromCML patients that wasand lysed as described [17].Purity was typically >98%as assessed by not seen in normal controls. This hybrid gene has elevated protein tyrosine kinase macia Biotech Inc.) waesquilibrated in 50 mM Tris,pH 8.0,50 mM NaCl activity and has been shown to causea CML-like syndrome in by repeated washing and a16mm x 100-cm column pouredT.he column mice [5, 6] and to transform immature hematopoietic cells in was washed extensively with lysis buffer, and theclarified K562 lysate was applied to the column at a flow rate of50mVh. The column was.
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